The sites of protein interaction on calmodulin (CaM) and the regulation of cyclic nucleotide phosphodiesterase (PDE) and calcineurin (CN) by CaM were investigated using chemical and enzymatic modification procedures and limited proteolysis. A calcium-dependent increase in the reaction between CaM and N-succinimidyl propionate (NSP) suggests exposure of two sites with increased reactivity located on CNBr fragment-2. Selective derivatization during and after immobilization on melittin-Sepharose (a new affinity matrix for purification of CaM) may permit isolation of a specific binding-protein domain on CaM. Proteolysis of PDE with chymotrypsin in the presence of EGTA yields a homodimer of 80-90k Da which cannot interact with CaM and is fully activated; in the presence of CaM, somewhat larger species are produced which are activated yet capable of interaction with CaM. A model is proposed which distinguishes specific functional domains for catalysis, CaM binding, and inhibitory control on PDE. High affinity antibodies react with two forms of PDE and its proteolytic fragments but show no reaction with CN or several other proteins. A method for covalent attachment of purified IgG to Protein-A Sepharose was developed providing an efficient procedure for analysis of PDE from different sources. The phosphatase activity of CN was compared using p-nitrophenyl phosphate (PNPP) and phosphotyrosyl glutamine synthetase (GSTP). The enzyme was dependent on Mn2+ for activity and was further stimulated 3- to 4-fold by CaM. While the Vmax was much higher for PNPP, the lower Km for GSTP (3 MuM vs. 25 mM) was consistent with a role for CN as a protein phosphatase. Phosphatase time-dependence showed an unusual activation-deactivation phenomenon observed only with CaM. Chymotryptic cleavage of CN produced a highly activated species which was inhibited by the addition of CaM. These data suggest a modulatory, rather than strictly stimulatory, role for CaM in regulation of phosphatase activity. Enzymatic carboxymethylation of PDE or CN suppressed CaM-dependent activity while having little effect on basal reaction rate. This selective effect on activity may allow studies on the mechanism and site of CaM-dependent regulation.